CFlm25 mediated alternative polyadenylation regulates fibrosis in systemic sclerosis

系统性硬化症（SSc）是一种多系统的纤维化疾病，其影响皮肤和多种内部病变。 机关持续性肌成纤维细胞活化和相关的过量细胞外基质蛋白（ECM） 沉积物是这种疾病的标志。导致这种过度纤维化反应的机制 仍然难以捉摸尽管其死亡率和发病率很高，但尚无FDA批准的治疗纤维化的药物 SSc的并发症我们最近的研究表明，一种被称为替代的RNA加工机制， 多聚腺苷酸化（阿帕）是通过3'非翻译区尾上调真皮纤维化中促纤维化基因的关键 缩短关键记录。最近的证据表明，阿帕的关键调节因子是切割因子Im 25（CFIm 25）。CFIm 25缺失导致3' UTR缩短。截短的3' UTR尾通常缺乏 microRNA结合位点并逃避microRNA介导的基因阻遏。我们的初步数据显示 CFlm 25在SSc患者的纤维化皮肤和鼠真皮纤维化模型中下调。敲低 正常皮肤成纤维细胞中的CFIm 25足以促进关键促纤维化基因的3' UTR缩短。 此外，中枢纤维化细胞因子TGFβ通过miR-203上调抑制CFIm 25表达。 总之，我们的数据揭示了TGFβ介导的CFIm 25下调导致3'端细胞凋亡的新机制。 UTR缩短和促纤维化因子和ECM的过度产生，并有助于发病机制 的SSc。基于这一初步数据，本项目的主要假设是TGFβ和miR-203 下调成纤维细胞中的CFIm 25，通过上调促纤维化基因导致真皮纤维化 和ECM通过3' UTR缩短。将在以下具体目标中审查这一假设： 目的I：确定CFIm 25耗竭在真皮纤维化鼠模型中的成纤维细胞特异性贡献。 这一目标将阐明CFIm 25耗竭对关键纤维化途径的下游影响。目标二： 确定CFIm 25下调的机制并评估其作为治疗药物的潜力 真皮纤维化的靶点。TGFβ介导的miR-203下调及后续的机制 将阐明miR-203对CFIm 25的抑制。这一目标将成为上游事件的特点， CFIm 25耗竭，并将确定潜在的治疗靶点。目的III：表征SSc人中的CFIm 25 皮肤/成纤维细胞和鉴定阿帕失调的纤维化基因。连续真皮成纤维细胞和皮肤 来自早期SSc患者和匹配对照的样本将使用一种新的RNA测序进行检查。 技术.这一目标将首次提供CFIm 25介导的APA的无偏见的纵向视图。 纤维化疾病的特征。 该研究首次将CFIm 25介导的3' UTR缩短与真皮纤维化联系起来。这 可以导致发现一个关键机制，放大SSc的纤维化反应，并最终 确定了一个全新的目标，用于治疗患有这种潜在破坏性疾病的人。

Systemic sclerosis (SSc) is a multi-system, fibrotic disease that affects the skin and a variety of internal organs. Persistent myofibroblast activation and associated excessive extracellular matrix protein (ECMs) deposition are hallmarks of the disease. The mechanisms that account for this excessive fibrotic response remain elusive. Despite its high mortality and morbidity, there are no FDA approved medications for fibrotic complications of SSc. Our recent work indicates that a RNA-processing mechanism known as alternative polyadenylation (APA) is critical for the upregulation of profibrotic genes in dermal fibrosis through 3’ UTR tail shortening of key transcripts. Recent evidence demonstrated that a key regulator of APA is Cleavage factor Im 25 (CFIm25). The CFIm25 deletion leads to 3’ UTR shortening. A truncated 3’ UTR tail will often lack microRNA binding sites and evades the microRNA-mediated gene repression. Our preliminary data indicate CFlm25 is downregulated in fibrotic skin of SSc patients and in murine dermal fibrosis models. Knockdown of CFIm25 in normal skin fibroblasts is sufficient to promote the 3’ UTR shortening of key profibrotic genes. Moreover, the central fibrotic cytokine TGFβ suppresses CFIm25 expression through miR-203 upregulation. Overall, our data uncovered a novel mechanism by which TGFβ mediated CFIm25 downregulation leads to 3’ UTR shortening and the over-production of profibrotic factors and ECMs, and contributes to the pathogenesis of SSc. Based on this preliminary data, the main hypothesis of this project is that TGFβ and miR-203 downregulate CFIm25 in fibroblasts, resulting in dermal fibrosis by upregulation of profibrotic genes and ECMs through 3’ UTR shortening. This hypothesis will be examined in the following specific aims: Aim I: Define the fibroblast specific contribution of CFIm25 depletion in dermal fibrosis murine models. This aim will elucidate the downstream effects of CFIm25 depletion on key fibrotic pathways. Aim II: Determine the mechanisms for CFIm25 downregulation and assess their potential as therapeutic targets in dermal fibrosis. The mechanisms for TGFβ mediated miR-203 downregulation and subsequent CFIm25 repression by miR-203 will be elucidated. This aim will characterize the upstream events leading to CFIm25 depletion and will identify potential therapeutic targets. Aim III: Characterize CFIm25 in SSc human skin/fibroblasts and identify fibrotic genes dysregulated by APA. Serial dermal fibroblasts and skin samples from patients with early SSc and matched controls will be examined using a novel RNA sequencing technology. This aim will provide for the first time an unbiased, longitudinal view of CFIm25 mediated APA profile in a fibrotic disease. The proposed research links for the first time the CFIm25 mediated 3’ UTR shortening to dermal fibrosis. This can lead to discovering a key mechanism that amplifies the fibrotic response in SSc and ultimately to identifying an entirely novel target for treatment of persons with this potentially devastating disease.